Project Summary Neuronal plasticity allows neurons to change the strength of their connections with each other and even to make or break connections. Plasticity is a fundamental property of neurons that underlies numerous brain functions such as learning and probably sleep, but it is also misregulated in diseases such as autism. Making stable changes in neuronal connections requires transcription and translation and an activity-dependent gene expression program is rapidly induced in response to neuronal activity. Many of the genes in this first wave of gene expression encode transcription factors that then regulate additional genes that are more directly involved in plasticity. Mutations in components of these activity-dependent programs have been associated with human cognitive disorders and psychiatric diseases, showing the importance of this pathway. We study plasticity in s-LNvs, the principal Drosophila circadian pacemaker neurons, which are ideal since changes in the morphology of their projections are predictable and happen at defined times each day. Having only 4 s-LNvs per brain hemisphere makes their projections easy to visualize, and we have the tools of Drosophila genetics to alter gene expression or neuronal activity in s-LNvs, along with expression profiles. s- LNv structural changes are driven by neuronal activity: their projections expand at dawn when s-LNvs are most excitable, and retract around dusk when s-LNvs become hyperpolarized. s-LNvs use activity-dependent gene expression to expand projections, ultimately activating Rac1 GTPase to regulate actin. We have identified a second transcriptional program that is activated by neuronal hyperpolarization and/or neuronal inactivity. This program opposes activity-dependent gene expression and leads to Rho1 GTPase activation to retract s-LNv projections. Just like activity-dependent gene expression, the first step in hyperpolarization-dependent gene expression is to transcribe a gene encoding a transcription factor ? in this case Toy, a fly Pax6 orthologue. In Goal 1, we propose to understand the molecular mechanism of hyperpolarization-dependent gene expression in s-LNvs, and test if this program functions in mammals. We will also test if hyperpolarization- dependent gene expression is important in sleep, which is associated with overall synaptic downscaling. In Goal 2, we will study competition between the activity-dependent and hyperpolarization-dependent gene expression programs that likely works both transcriptionally and post-transcriptionally to ensure one program dominates. In Goal 3, we will develop a genomic-based approach to identify connections between neurons that we predict will be broadly applicable, and also to give insights into how new connections are specified at the molecular level. Overall, studying plasticity in s-LNvs should give a holistic view of plasticity that is broadly relevant across neurobiology and could identify new disease risk loci.